Early cardiac cushions are located in the atrioventricular canal (AV) of the embryonic heart. These cushions are populated with mesenchymal cells and are the primordia for the cardiac valves and membranous septa. A great deal of progress has been made in identification of genes that regulate the initial steps of cushion brmation (stage14-22) (Gitleretal., 2003;Moorman and Christoffels, 2004). However, little is known about how these cushions differentiate into valve leaflets (stage 35-45). As valvular defects are among the most common and deleterious of all cardiac malformations, it is critical that the mechanisms of late valve development be delineated as well. Researchers studying early valve development have benefited from an excellent in vitro model of the initial stages of cushion formation. Until recently, such a model for late stage valve development has not existed. The proposed research uses an in vitro model we have developed to study late valve leaflet formation. This new model is composed of a collagen tube scaffold in which cushion anlage matures into valve tissue (Goodwin et al, 2005). This new model expands upon the previous cardiac cushion model (Runyan and Markwald, 1983) by providing a three-dimensional (3-D) environment in which cardiac cushion tissues recapitulate the later stages of valve development, both at the molecular and morphological levels. Initial experiments testing this model indicate that valve leaflet morphogenesis and extracellular matrix (ECM) protein expression/deposition is dependent on fluid flow in the tube model. The central hypothesis being tested in this proposal is that fluid flow plays a key role in valve leaflet morphogenesis. The tubular model system has the unique ability to test the role that fluid flow plays in valve morphogenesis. To address the hypothesis of this application, we will pursue three specific aims:Aim 1) Determine the effect of fluid flow on the differentiation and morphogenesis of valve leaflets. Aim 2) Determine the effect of fasciclin missexpression in flow-regulated valve cultures. Aim 3) Determine the role that proepicardial (PE) cells have on AV cushion maturation. This proposal is designed to provide specific data that describe the flow conditions that sustain the early stages of valve leaflet formation. This information would be valuable for the design of tissue-engineered valves as well as to provide new strategies for the treatment of birth defects.